JPH01503410A - Amperometric method for the determination of 1,4-dihydronicotinamide adenine dinucleotide (NADH) in solution - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] 1,4-dihydronicotinamide adenine dinucleotide (NADH) in solution Amperometric method for the determination of 1,4-dihydronicotin in solution Amperometric method for the determination of amide adenine dinucleotide (NADH) Regarding the law.

NADH and its oxidized partner NAD undergo redox catalyzed by numerous enzymes. It is a cofactor in the gas reaction. In some cases, the enzyme's substrate is a cofactor. oxidation in the presence of vector NAD and a suitable oxidase or dehydrogenase. in solution to produce NADH; in other cases, the enzyme's substrate is The factor is reduced in the presence of NADH to form NAD in solution. Many In many cases, the determination of NADH concentration, as an indicator of substrate concentration, Or as a means to track the process of enzymatic reactions involving NADH (or NAD). can be used.

The concentration of NADH in solution can be measured colorimetrically, but colorimetric methods are generally inaccurate. It is known to be beneficial. Electrochemical methods are even more disadvantageous, but NAD Attempts to determine H electrochemically have so far been successful to a very large extent;

For example, the concentration of NADH can be determined by an amperometric assay. It is known that NADH is immobilized at an electrode and controlled to The current passing under appropriate conditions is proportional to the concentration of NADH. No Advantageously, electrochemical oxidation of NADH requires high potentials and DH is generally not oxidized cleanly on the electrode surface; for example, in the case of The surface of the electrode quickly becomes contaminated due to the formation of a film on the surface, which is caused by electrification. Affecting the magnitude and speed of the scientific response: 1. Moirou x) and P, F. Erping (E I v i n g), Journal O. American Chemical Society (J, Am, Chem.

Soc, ) (1980) 102.6533-6538, and G.

Johnson: LM, D, Ryan and G.

S. Wilson, Analytical Chemistry , Chem, ) (1986) 58.42R0 Many efforts have been made to avoid these problems. Attempts have been made. For example, a modified electrode coated with a layer of conductive organic salts Suggested use: J. J. Kulys, Biosensors. (Bios e n5ors) (1986) 2.3-13° or Redox mediators such as Meldora Blue can be used to more effectively couple the oxidation reaction to the electrode and/or It has been proposed to lower the potential: L. Gorton et al. Le Electroanalytical Chemistry (J, EIelectroanaly t, Chem, ) (1984), Udan, Sat. Dox mediators have been used in free solutions. Ta. For example, methoxyphenazine methosulfate is a modified pyrolytic graphite electrode. Has been used with poles: Y.

Kimura and Nihi, Analytical Science Analytical 5 Sciences (1985), L 271 −4°Other experiments used palladium, graphite, and glassy carbon electrodes. However, there is still no way to quickly and reproducibly determine NADH. Electrochemical methods for determining this have not yet been developed.

According to the invention, NADH has an excellent amperometric response; Both a buffer containing H and a solution containing an enzyme, a substrate for the enzyme and NADH. Researchers have discovered that it is possible to oxidize cleanly using activated carbon electrodes. and the activated carbon electrode is of the type used in fuel cell technology. , and a natural or synthetic resin binder, preferably a synthetic hydrophobic binder. , such as a fluorocarbon resin, most preferably polytetrafluoroethylene preferably platinated or palladized (these terms refer to When used herein, may contain oxides of platinum and/or palladium. materials containing platinum or palladium metals; particles of carbon or graphite (including materials treated with or treated with carbon or graphite) Consisting of a heterogeneous resin bonded layer of precious metals containing metal particles. Preferably platinized; uses palladized carbon or graphite particles, where carbon or are graphite particles, colloidal platinum or palladium metal particles, Platinum or palladium geode is adsorbed onto the surface of the powdered particles before bonding. The resulting electrode is platinized or palladized by precipitation. do-like platinum or palladium, or the corresponding oxide, uniformly distributed throughout the layer, Consists of a layer of heterogeneous porous activated carbon powder. platinized or palladized Resin bonded layer of activated carbon or graphite particles is self-supporting but usually a support member, preferably an electrically conductive support member, preferably platinized or palladized. conductive carbon in which particles of carbonized carbon or graphite are bonded as a surface layer supported by a base paper or said carbon or graphite particles are carbon Impregnated into the web of fibers. Platinized or palladized materials are preferred However, other noble metal-containing activated carbon electrodes can be used, e.g. gold-containing electrodes. Ru.

Here, the terms "platination" and "palladation" refer to oxides unless otherwise specified. include.

Also herein, the terms "activated" carbon, "activated" graphite, etc. "More than 7g, more usually more than 200m'/g, e.g. 200-600m"7 Highly porous, high surface area carbon and graphite, with surface areas greater than or equal to g Mention the material. Materials with such surface area may be carbon or graphite, for example. In this field, the powder of It is obtained by producing a high surface area product called carbon dioxide.

Very apart from the stability, reproducibility and rapid response already mentioned, the material of the invention A further particular advantage of using them is that they reduce the concentration of NADH at relatively low potentials. , e.g. in the range 0-600 mV or even at negative potentials. For example, for monitoring the concentration of NADH using glassy carbon or graphite electrodes. This means that it is possible to monitor the required voltage of 750 mV or more. child Thus, the electrode of the present invention has a relatively low background current and therefore an improved It is characterized by its sensitivity. Electrodes can also be used in biological or clinical samples. low response to potential interfering species, e.g. uric acid, frequently present in characterized by

Preferred electrode supports for use in accordance with the present invention are, in fact, by Protech Company in the Highlands. and is conventionally used as an electrocatalytic gas diffusion electrode in fuel cells. is a commercially available material. Such materials are covered by US patents (U. S-A) No. 4.044,193, United States Patent (US-A) No. 4.166.143 No. 4.293.396 and United States Patent (US-A) No. 4.478°696, please refer to those documents for details. death However, in large details, 15-25 angstroms (1,5-2°5 Colloidal platinum with a particle size in the range of nanometers (nm) is mixed with powdered carbon (particle size 50 For example, a platinum sol is placed on a surface of ~300 angstroms (5~30 nm). Formation in situ in the presence of powdered carbon that acts as a nucleating agent for the sol to adsorb it. The platinized carbon particles are then placed on a conductive support structure. , for example, a synthetic resin binder, preferably a fluorocarbon resin, on a conductive carbon paper. , in particular using polytetrafluoroethylene. Or similar particles Platinum or palladium oxides with a range of particle sizes can be substituted for colloidal platinum. and adsorbed onto carbon or graphite particles in a similar manner. You can wear it.

In the alternative method described in United States Patent (US-A) No. 4.293.396, The platinized carbon particles are impregnated into a preformed porous carbon cloth, and then the platinized carbon particles are impregnated into a Therein using orocarbon resin, preferably polytetrafluoroethylene Join. However, the invention is not limited to the use of platinized or platinized materials. or palladized; or other noble metal-containing activated carbon or graphite; Comprising other similar support materials consisting of a porous resin bonded layer of particles of phytophyte .

Platinized carbon or graphite particles are used to bind palladized carbon or graphite particles. Preferred resin binders used for this purpose are hydrophobic fluorocarbon resins, especially polytetra trifluoroethylene but other suitable natural or synthetic resin binders; For example, polyethyl methyl acrylate, polyvinyl acetate, polyvinyl chloride, polyvinyl chloride, Recarbonate, poly(4-methylpentene-1) polyisoprene, polychloro Contains prene, boilie (1,3-butadiene), silicone rubber and gelatin can.

The weight ratio of binder to noble metal-containing activated carbon or graphite particles is: 10-75% binder and 90-25% activated carbon or graphite and preferably 20-50% binder and correspondingly 80-50% activation. carbon or graphite. Precious metals, e.g. white activated gold or palladium or their corresponding oxides, or gold, The amount of carbon or graphite added to the particles is 1-10%, preferably 2-8%, maximum based on the total weight of light and binder. It is also preferably 4 to 6%.

Resin/activated platinized or palladized carbon powder directly onto a suitable support , for example, instead of attaching directly onto the surface of conductive carbon particles, a binder and white A mixture of gold-plated or palladized carbon powder is suspended in a suitable inert medium. and applied by screen printing technique to the surface of the support, thereby supporting Platinized or palladized resin bonded onto the surface of the carrier; It is possible to form a thin film.

As well as direct quantitative measurement of NADH in solution, the electrodes and methods of the present invention , in the quantitative measurement of NADH generated or consumed in situ, e.g. and its cofactors. child For example, reactions such as lactate dehydrogenase lactate dehydrogenase Conversion to tate, i.e., reaction lactate + NAD (This reaction can be monitored by the decrease in the concentration of NADH) Conocton Ten NADH (This can be monitored by the increase in the concentration of NADH as the reaction progresses. oxidation of glucose to gluconolactone by for this purpose , the activated platinized or palladized carbon electrode used according to the invention is By enzyme immobilization techniques known in this field and, for example, in Europe. As taught in patent application (EP-A) O'247850, resin-bonded carbon Enzymes incorporated into or immobilized on the substrate, e.g. can have todehydrogenase or glucose dehydrogenase .

In a further variation of this concept, the present invention also provides single-use disposable enzymes. electrodes and methods, in which the enzyme electrode itself is only an immobilized enzyme; and also a suitable cofactor for the enzyme, optionally NAD; is composed of NADH, and thus the enzyme electrode contains the relevant substrate-containing When in contact with a sample that Since the cofactor is supplied by the electrode itself, it is possible to ensure that the sample contains the cofactor. enzyme, as determined by changes in the concentration of NADH, regardless of whether A cophasis of NAD or NAD)I with the ability to amperometrically respond to the activity of The vector may include, for example, NAD or NAD in any suitable manner in the electrode. Incorporation can be done by impregnation with any solution of H and drying.

As is well known in this field, the surface of the electrode material is a porous membrane, e.g. For example, protected by a polycarbonate film with a pore size of approximately 0.03 μm. It can be protected or not protected. Other suitable membrane materials can also be used. Wear.

The invention will be further described with reference to the accompanying drawings.

FIG. 1 shows a sample used to test the NADH response of an activated carbon electrode according to the present invention. The modified Rank brothers electrochemical FIG. 3 is a diagrammatic cross-sectional view of a cell.

FIG. 2 shows a NADH cell using platinized carbon paper (PCP) electrodes according to the present invention. Figure 2 shows the response of the electrode to sequential additions to

Figure 3 shows the relationship between NADH at various balance potential pairs Ag/AgC1 reference electrode. The response of the PCP electrode is shown below.

Figure 4 shows pyruvate in the presence of lactate dehydrogenase (LDH). shows the response of the electrode to

Figure 5 shows acetal in the presence of alcohol dehydrogenase (ADH). The response of the electrode to dehyde is shown.

FIG. 6 shows the response of a platinized carbon paper electrode to the concentration of NADH according to the present invention. This is another graph.

FIG. 7 shows the results of another experiment involving the response of the electrode to pyruvate.

Figure 8 shows the enzymatic oxidation of glucose using glucose dehydrogenase. shows the response of the electrode to in situ generated NADH.

FIG. 9 shows a similar response curve for a platinum oxide containing electrode.

Figure 1O shows the response curve for a palladized activated carbon electrode.

In the following examples, various platinized or palladized carbon papers (PCP ) Rank oxygen electrode system with modified electrode [Rank Brothers (Rank B Rothers), Cambridge, Bocchijam]. These responses were tested and as shown in the accompanying drawing (FIG. 1). correction The system of ranked cells consists of a two part cell with base (1) and It consists of an annular jacket (2) surrounding a water chamber (h), said jacket The temperature of the cell can be controlled by circulating water through the connected together by a captive threaded collar (3) Ru. A platinum contact button (d) is located in the center of the base (1); Above is placed a test disk (a) of low electrode material, and this disk ( a) The rubber O-ring seals when the two parts of the cell are joined together held in place on the platinum contact button (cl) by (e) and (f) .

The cell naturally contains the NADH-containing substance to be tested, and a stopper (4) is inserted into the top of the cell. This is supported by a suitable collar (g) and has a platinum contact electrode therein. A pole (b) and an Ag/AgC1 reference electrode (c) are attached. The exam is Balanced at various potentials ranging from 100 to 600 mV with reference to the Ag/AgC+ electrode. Perform the test using the appropriate electrode. Other tests are European patent publication @ (EP-A) 0 The two illustrated in and detailed in Figure 16 of No. 247-850. The test was carried out in a cell with two electrodes. In a two electrode cell embodiment, one of the electrodes is The material is polycarbonate (for platinum contact buttons at the base of the cell) 0.03 μm pore size) and to which the NADH-containing sample was applied. The annular Ag/AgC1 reference electrode is connected to the platinum contact at the base of the cell. around it, but separated from it by an insulating sleeve. electrode cell is polarized at various potentials with respect to the Ag/AgC1 electrode, and the output current is Monitor at various potentials.

The invention is illustrated by the following examples. In this embodiment, the potential material is Protech Company, Newton Highlands, Satsut. Company) and developed as a gas diffusion electrode, It is platinized carbon paper (PCP). PCPK polar material is covered by United States Patent (US-A) No. 4.044.・Carbon powder particles (Vulcan XC- 72)% nominal particle size 30 nm) using H,O, in the presence of carbon powder The complex salt platinum sulfite is oxidatively decomposed, thereby forming a colloid on the surface of carbon powder particles. The platinum was first prepared by precipitating platinum, particle size 1.5-2.5 nm. become After platinization, the platinized carbon powder is subsequently used for commercial graphitization. Approximately 50% by weight of polytetra based on platinized carbon powder is deposited on the surface of the electrically conductive carbon paper. Shape and bond using trifluoroethylene as a bonding agent. can get The platinized carbon paper electrode material has a thickness ranging from 0.1 to 0.5 mm and a thickness of 0.24 m. It has a platinum content of "g/am".

For the purpose of the following tests (Examples 1-3), carbon paper electrode material was on the platinized electrodes of the cell system shown in Figure 1 of the accompanying drawings. attached to. Actual area of the carbon paper electrode exposed to the sample in each case The height is 0-16 cm.

The results obtained are as follows: Example 1 Electrochemical oxidation of NADH on platinized carbon paper (PCP) using standard potentiostatic technique a standard sample of 20 mmol NADH in Tris/HCI pH 9 buffer. , 2 mm of a cell containing 0.1 molar pH 7 phosphate/1 molar KCI buffer. Add it to the bottle. Platinized carbon paper electrode (P　CP) as Ag/AgC] reference electrode were balanced at various potentials. The counter electrode was platinum. stable current A plateau of t was obtained (Fig. 2), which is proportional to the concentration of NADH (Table 2). 1 and 3).

Q for NADH at various equilibrium potentials, 95 7 15 28 1.8 13 25 53 2.7 - 32 73 4.0 38 42 113 6.6 - 52 183 10.0 60 - 1 Example 2 NAD to pyruvate in the presence of lactate dehydrogenase (LDH) H-electrode system response 12.5 mmol NADH in 2 ml pH 7 phosphate/KCI buffer and A similar cell was constructed containing pyruvate and 10 mmol of pyruvate. Use opposite voltage The pole and reference electrode were as in Example 1. The electrode used is 400m Balanced at V and a constant signal of 170 μA above background gave. When 120 units of LDH (bovine heart type xv) is added, the current is 92 The enzymatic conversion of pyruvate to lactate decreases at an initial rate of μA/min (Fig. 4). We show that the oxidation can be efficiently monitored by NADH electrochemically coupled to the electrode. be done.

Acetalde in the presence of alcohol dehydrogenase (ADH) on the PCP electrode Response of the NADH-electrode system to 2 ml pH 9 phosphate/KCI Contains 3 mmol NADH and 35 mmol acetaldehyde in buffer I assembled a cell to do this.

The counter and reference electrodes used were as in Example 1.

The electrodes used were balanced at 400 mV and 40 mV above background. A constant signal of 0 μA was given. When 2 units of ADH (Ua liver) are added, the current is Acetonitrile to ethanol decreases at an initial rate of 130 μA/min (Figure 5). The enzymatic conversion of is efficiently monitored by NADH electrochemically coupled to the electrode. It is shown that

In the following examples, the steric arrangement of two electrode cells or three electrode cells It was used. A three electrode cell is described herein and illustrated in FIG.

A two-electrode cell is shown in Figure 16 of European Patent Application (EP-A) No. 0247-850. It has the same configuration as shown and the reader is referred to this patent for details. sea bream.

Example 4 (Figure 6) Data was collected using a two electrode configuration polarized at 200 mV.

16 mmol/Q N a H2p O4% 53 mmol/12 Na2HP O,, 52 mmol Q of NaC 1% 1.5 mmol/Q of ethylenediamine tetra Acetic acid, pH 7.4, was used. Stable background current in this buffer After achieving this, the buffer is blotted onto the membrane and a sample of NADH in the same buffer is Replaced with Figure 6 shows commercially available platinized carbon from Prochik Company. shows the response from the paper electrode, and this carbon paper is placed on a support sheet of conductive carbon paper. The platinized carbon layer consists of precipitated resin-bonded platinized carbon particles. , by weight, 50% polytetrafluoroethylene, 45% fine carbon (V ulcan XC72) and 5% colloidal platinum preadsorbed onto carbon powder Consisting of

To minimize background current, a 5 mg/ml protein solution ( Glucose oxidase) was adsorbed onto the electrode overnight before NADH measurement. gluco It should be understood that -sugar oxidase is just one example of a suitable protein. .

Example 5 (Figure 7) This illustrates the applicability of the invention to the determination of substrates for NADH-utilizing enzymes. 3 A two electrode cell was used as previously described; but equipped with a magnetic stirring bar. Electricity used The electrodes were platinized carbon paper as in Example 4, but L-lactate dehydrocarbon Genase (EC1,1,1°27 from beef heart) by binding to carposiimide. and immobilized on the electrode, see European Patent Application (EP-A) No. 0 247 850, but Lactate dehydrogenase [Sigma Chemi cals), type XV, 50 units/mg of protein] lmg/m A solution of No. 1 was used.

The cell is Q, l mo/l, /12 (7) phosphate/1-f nyl/Q no K Initially contained 12.5 mmol/Q NADH in CI buffer, pH 7. . The platinization potential was 350 mV. Using this equipment, as shown in the drawing, Consumption of NADH was monitored when an aliquot of pyruvate was added to the cell.

Example 6 (Figure 8) Example 5 was repeated, but instead of stock immobilized lactate dehydrogenase Glucose dehydrogenase (Baciflus ssp) immobilized on the electrode EC1,1,1,47 from Sigma, 100-300U/m g of protein) was used in a similar manner.

Consumption of NADH using lactate dehydrogenase pyruvate ten NADH → Pyruvate was measured by monitoring lactate + NAD, but glucose Using course dehydrogenase to produce NADH: β-D-glucose +NAD → D-gluconolactone + Glucose was measured by NADH . Cell is 0.1 mol/Q phosphate 10° 1 mol/Q KCI/2.4 mmol /Q NAD, pH 7.

Example 7 (Figure 9) Following the procedure outlined in Example 4, the current output of the platinum oxide-containing carbon electrode was 200 mV (Ag/ AgC+ reference electrode) and equal to that of the platinized carbon paper electrode. shows a virtually linear response. In this case, the electrode material is resin bonded (polyte Consists of a layer of #l elementary particles (Vulcan XC72), This layer consists of 5% by weight (based on the total weight of resin-bound particles) preadsorbed onto the carbon powder particles. ): 50% by weight binder, 45% by weight carbon, and conductive tray (To ra'y) (trademark) carbon paper surface.

Example 8 (Figure 1O) Once again, following the procedure outlined in Example 4, the platinized carbon paper electrodes were Various concentrations of NADH at an outflow power of 200 mv (relative to Ag/AgC1) Measurements were made using a two-electrode cell. Once again, the response (Figure 10) is It is qualitatively linear. The electrode material was as described in Example 7, except that resin The bonded carbon particles consist of 5% by weight of preadsorbed fine palladium.

The above examples show that the electrode materials were used to produce rapid and reproducible oxidation of NADH. prove the use of These responses are generally slow and relatively insensitive. and very poorly reproducible (with rare exceptions) platinum, glassy carbon, or as demonstrated in comparable experiments using graphite electrode materials. , which is in marked contrast to that provided by most other electrode materials. circle The effectiveness of the platinized or palladized carbon electrodes we used specific heterogeneous structures of and its association with biological molecules such as NADH and enzymes. It appears to be a result of conformity. Oxidation of NADH is also caused by enzymes and substrates. for a rapid NADH binding enzymatic assay that proceeds efficiently in the presence of Can be used as a reference. Pyruvate (using LDH) and acetate The possibility of an efficient assay for toaldehyde (using ADH) is limited by the above implementation. As is clear from the examples, a large number of similar assays are also available for other enzymes and This can be achieved using a substrate.

Although the invention has been described primarily with reference to the determination of NADH in solution, phosphorylated 1,4-dihydronicotinamide adenine dinucleotide (NADPH), Thus, phosphorylated NADH can be determined by exactly the same technique. So Moreover, NADPH (or NADP) rather than NADH (or NAD) Because they are cofactors in a selected group of enzyme-catalyzed reactions, The technique works in exactly the same way, i.e. the consumption of NADPH in solution or can be monitored by determining amperometrically any of the production shall be.

Thus, all references to NA, DH or NAD, unless otherwise specified, are , should be construed to include phosphorylated derivatives.

Steady state current J, lA LDH in l2O time seconds Millimoles/jNAD Concentration of pyruvate mmol/,! ! Glucose concentration mmol/2 0 10 20 3O NADI-1 mmol/2 NAO) l mmol/2 international search report international search report Koyo 8ε0033a SA　22049

Claims (1)

[Claims] 1. Bringing the NADH-containing solution into contact with an activated carbon electrode, maintaining the carbon electrode at a controlled fixed potential effective to cause oxidation of DH; and measuring the current output from said carbon electrode, where the noble metal containing An activated carbon electrode is used, and the activated carbon electrode is made of activated carbon or graphite. pre-adsorbed onto the particles of phytochemicals with finely divided noble metals or corresponding oxides. , and activated carbon combined with a natural or synthetic resin binder. Characterized by consisting of a porous, heterogeneous resin-bonded layer of bare or graphite particles 1,4-dihydronicotinamide adenine dinucleotide (N A method for quantifying ADH). 2. The method according to claim 1, wherein the resin binder is a fluorocarbon resin. . 3. The claim that the fluorocarbon resin is polytetrafluoroethylene. The method described in Section 2. 4. The resin-bonded noble metal-containing carbon or graphite particles are bonded to the underlying support. Any one of claims 1 to 3, which is formed as a resin bonded surface layer on the member. The method described in. 5. The method of claim 4, wherein said underlying support member is electrically conductive. 6. The conductive support member is a layer of conductive carbon paper, and the noble metal-containing carbon is applied to this layer. or according to claim 5, wherein graphite particles are bonded as a surface layer. Method. 7. The resin-bound precious metal-containing carbon or graphite particles are contained in the carbon fiber web. Any of claims 1 to 3 impregnated with and supported by Method described. 8. The noble metal-containing carbon or graphite particles have a particle size in the range of 5 to 30 nm. 8. The method according to any one of claims 1 to 7, comprising: 9. The carbon or graphite particles are preplatinated or prepalladized. particles preadsorbed onto graphite or carbon particles; Claims 1 to 1 containing finely divided platinum or palladium or a corresponding oxide. The method described in any of Section 7. 10. Using a platinized or palladized carbon electrode, the carbon electrode is as preplatinated or prepalladized carbon or graphite particles; of colloidal platinum or palladium metal preadsorbed on their surfaces. particles, said colloidal platinum or palladium particles having a diameter of 1.5 to 2.5 nm. 9. The method of claim 8, having a range of particle sizes. 11. NADH or as a cofactor in the reaction between an enzyme and its substrate Claims that apply when monitoring the production or consumption of NADH, including any NAD The method according to any one of items 1 to 8. 12. A noble metal having the enzyme incorporated or immobilized thereon or therein. A carbon-containing electrode is used, and the electrode activates the enzyme by changing the NADH concentration. used to monitor the activity of the enzyme involved in the reaction between the enzyme and its substrate. containing the enzyme substrate in the presence of either NAD or NADH as a cofactor. 12. The method according to claim 11, wherein the electrode is brought into contact with a sample containing the electrode. 13. If appropriate, in combination with NADH or NAD cofactor, the enzyme Precious metal-containing material having an element incorporated or immobilized thereon or in it using an electrode, and thus NADH or NAD cofactor is passed through the electrode into the sample. 13. A method according to claim 12, in which the method of claim 12 is introduced. 14, consisting of an activated carbon electrode, said electrode being made of activated carbon or graphite. with finely divided precious metals or corresponding oxides preadsorbed on the particles of metal, and activated carbon or bonded with a natural or synthetic resin binder. consists of or consists of a porous, heterogeneous, resin-bonded layer of graphite particles; The resin bonding layer has enzymes incorporated or immobilized thereon or in it. in combination with NAD or NADH as a cofactor of said enzyme; said electrode in the presence of a substrate for said enzyme and optionally said NAD or amperometrically responsive to the activity of said enzyme in the presence of NADH. 14. A single-use disposable enzyme electrode for use in the method of paragraph 13. 15. The activated carbon electrode is bonded with and underlying fluorocarbon resin. supported on a support member and having said enzyme and said cofactor also thereon; has a preplatinated or prepalladized activation material immobilized therein. The material consists of a porous resin-bound heterogeneous surface layer of carbon or graphite particles. A single-use disposable enzyme electrode according to item 14. 16. The preplatinated or prepalladized activated carbon or graphite The underlying support member for the resin bonded layer of graphite particles is a conductive carbon paper 16. A single-use disposable enzyme electrode according to claim 15.